The last few years have seen the discovery of a new class of proteases that cleave the transmembrane domain of their substrates and that apparently contain a membrane-embedded active site. These intramembrane proteases are remarkably conserved and play many important roles in biology and human disease. One family of this class is distantly related to the presenilins and is exemplified by signal peptide peptidase (SPP), which cleaves remnant signal peptide fragments left in the membrane after the action of signal peptidase. SPP is critical for immune surveillance and is essential for the maturation of the core protein of the hepatitis C virus, suggesting it may be a worthy target for antiviral drug design. A putative aspartyl protease, SPP appears to share many biochemical properties with the presenilin-containing y- secretase complex. However, unlike presenilins, SPP apparently does not need to assemble with other protein cofactors nor is it processed into two pieces during its maturation into an active protease. In this application, we propose to work toward a mechanistic and structural understanding of SPP as a representative intramembrane protease, an emerging enzyme class of critical importance in biology and medicine. The detailed understanding of an SPP-type protease should provide tremendous insight into the mechanism of intramembrane proteolysis. The following specific aims are proposed: (1) How does SPP process substrates? We will address whether SPP cleave its substrates at two different sites, as does y- secretase, and whether SPP, like y-secretase contains an initial substrate docking site. (2) How are monomeric units of SPP arranged within a dimer? Using mutagenesis and oxidative crosslinking, we will determine which cysteines in SPP contribute to the dinner interface and how the transmembrane aspartates are aligned within the dimer. (3) What is the nature of the SPP active site? We have discovered that the C- terminal half of SPP alone is catalytically active;we seek to biochemically characterize this catalytic domain and work toward its structure by NMR. (4) What are the characteristics of SPP orthologs from extremophilic archaea? SPP-like proteases from extremophilic archaebacteria should be more stable and amenable to structural studies than other full-length SPPs. We will work toward expression, purification, and characterization of archaeal SPPs to set the stage for structural elucidation. LAY SUMMARY: Signal peptide peptidase (SPP) is a protein-cutting enzyme that plays an important role in the immune system, and the hepatitis C virus uses human SPP as part of its infection process. SPP is also related to a much more complicated enzyme that plays a key role in Alzheimer's disease and is widely considered an important potential target for therapy. The plan is to more fully understand how SPP works and to take steps toward purifying SPP for eventual elucidation of its structure. A structure of SPP would provide tremendous insight into the workings of an unusual new class of enzymes that are critical to both biology and medicine.